A: Infrared Absorption á197Kñ.

B: Utraviolet Absorption á197Uñ—

Test solution: 4mg per mL,in colorless pyridine from a freshly opened container.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (1:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Prepare a solution of ethylparaben in a mixture of water and acetonitrile (1:1)containing about 0.5mg per mL.

Standard preparation— Transfer about 10mg of USP Ethinyl Estradiol RS,accurately weighed,to a 50-mLvolumetric flask,and add 10mLof Mobile phase.Add 5.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.2mg of USP Ethinyl Estradiol RSper mL.

Assay preparation— Transfer about 25mg of Ethinyl Estradiol,accurately weighed,to a 25-mLvolumetric flask,add Mobile phaseto volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,add 5.0mLto Internal standard solution,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the resolution,R,between the analyte and internal standard peaks is not less than 4.5,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.6for ethylparaben and 1.0for ethinyl estradiol.Calculate the quantity,in mg,of C20H24O2in the portion of Ethinyl Estradiol taken by the formula: in which Cis the concentration,in mg per mL,of USP Ethinyl Estradiol RSin the Standard preparation,and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.